Biochemical investigations on bacterial and fungal dimethylallyltryptophan synthases

Prenyl transfer reactions occur ubiquitously in nature and play an important role in primary and secondary metabolism in all domains of life. Prenylated secondary metabolites including indole alkaloids usually demonstrate improved biological and pharmacological activities and thus makes them to prom...

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1. Verfasser: Winkelblech, Julia
Beteiligte: Li, Shu-Ming (Prof.Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2016
Pharmazeutische Biologie
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Zusammenfassung:Prenyl transfer reactions occur ubiquitously in nature and play an important role in primary and secondary metabolism in all domains of life. Prenylated secondary metabolites including indole alkaloids usually demonstrate improved biological and pharmacological activities and thus makes them to promising candidates for drug discovery and development. Important producers of such bioactive compounds are fungi of ascomycetes and bacteria of actinomycetes. The transfer reactions of a prenyl moiety from prenyl diphosphate, primarily dimethylallyl diphosphate (DMAPP), onto indole derivatives including tryptophan are mainly catalyzed in nature by the members of the dimethylallyltryptophan synthase (DMATS) superfamily. In the last years, remarkable progress has been achieved in their biochemical, molecular biological, and structural characterization, especially for DMATS enzymes from fungi. The major challenge of this thesis is to provide a better understanding of the catalytic features of these enzymes from different origins. Cloning and expression of these genes as well as the subsequent biochemical investigations of the three novel tryptophan prenyltransferases provided several new intriguing features. Initially, two tryptophan C6-prenyltransferases were identified, i.e. 6-DMATSSa from Streptomyces ambofaciens and 6-DMATSSv from Streptomyces violaceusniger. Biochemical investigation on these enzymes revealed a remarkable broad substrate specificity. In addition to a number of indole derivatives also several hydroxynaphthalenes were accepted by 6-DMATSSa and 6-DMATSSv. Moreover, they represent the first examples of tryptophan prenyltransferases that accept both DMAPP and geranyl diphosphate (GPP) as prenyl donors and catalyze the same prenylation positions. Consequently, the studied 6-DMATSs were used for further investigations on the acceptance of unnatural alkyl or benzyl donors. Prior to this study, investigations on this issue are limited to fungal C4- and C5- prenyltransferases. The L-tyrosine prenyltransferase TyrPT with a tryptophan C7-prenyltransferase activity, the two mentioned 6-DMATSs as well as the bacterial 5-DMATSSc (SCO7467) were included in this project. In total, five C5-, C6- and C7-prenytransferases were investigated in the presence of the DMAPP analogs methylallyl (MAPP), 2-pentenyl (2-pentenyl-PP) and benzyl diphosphate (benzyl-PP). The unnatural donors were accepted by all tested enzymes with different relative activities and regioselectivities. C6-alkylated or benzylated derivatives were identified in all the reactions, as unique product of the two 6-DMATSs or as one of the main products of the other enzymes. These results demonstrated a clear preference of the five enzymes for alkylation/benzylation at C-6 of the indole ring in the presence of the unnatural DMAPP derivatives. Furthermore, homology modeling of the 5-DMATS and subsequent docking as well as molecular dynamics studies with DMAPP, MAPP and 2-pentenyl-PP, led to a distance-based explanation of the observed reaction results. Later on, a third tryptophan C6-prenyltransferase 6-DMATSMo from Micromonospora olivasterospora was identified and characterized. The most notable feature of 6-DMATSMo is the high relative activity toward D-tryptophan. This result led to the comparative study on enantioselectivity of the seven DMATS enzymes from fungi and bacteria. The tested prenyltransferases displayed different substrate preferences as well as different regioselectivities toward the L- and D-enantiomers of tryptophan and their methylated derivatives. Interestingly, the bacterial 5-DMATSSc and 6-DMATSMo highly preferred the D-enantiomer of 5-methyltryptophan to the L-enantiomer, although that was the better substrate in all other reactions. In the presence of the racemate, the D-enantiomer reaction was strongly inhibited, which could be explained by the high affinity to the respective L-form as the main reason. Another interesting output of this project is the reduced or even completely changed regioselectivity for the reactions of FgaPT2, 5-DMATSSc, and 7-DMATS with the D-enantiomers of tryptophan or 5-methyltryptophan. Moreover, the observed diprenylation by 5-DMATSSc was the first report on a tryptophan prenyltransferase which catalyzes two successive prenylation steps. In conclusion, identification and characterization of the three new 6-DMATSs from actinomycetes expand our knowledge on bacterial tryptophan prenyltransferases. Furthermore, prenyltransferases including the three 6-DMATSs could serve as valuable biocatalysts in chemoenzymatic synthesis for alkylated compounds with potential biological activities.
Beschreibung:240 pages.
DOI:https://doi.org/10.17192/z2016.0790